Abstract
Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson’s disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.
Highlights
Parkinson’s Disease (PD) is a neurodegenerative disease of aging, characterized neurologically by uncontrolled tremors and bradykinesia due to loss of dopamine-producing neurons in the substantia nigra [1]
To rule out the possibility that VH14PEST binds to epitopes that are present only in α-Syn~GFP, we tested two additional paradigms
WT α-Syn was co-transfected with VH14 constructs into ST14A cells
Summary
Parkinson’s Disease (PD) is a neurodegenerative disease of aging, characterized neurologically by uncontrolled tremors and bradykinesia due to loss of dopamine-producing neurons in the substantia nigra [1]. Lewy bodies and Lewy neurites, containing misfolded aggregated α-Synuclein (α-Syn), are the most prominent neuropathologic finding [2,3,4]. Oligomeric, protofibrillar and fibrillar isoforms and multimeric structures can be found both within, and extruded from, affected cells. None of these can proceed in the absence of the primary intracellular α-Syn misfolding event, which is an important therapeutic target. The nonamyloid component (NAC) hydrophobic interaction region of α-Syn is critical for aggregation.
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